Isolation and characterization of a cadmium binding protein from the planarian, Dugesia dorotocephala, and evaluation of its protective effects against DNA damage caused by cadmium and radiation

Mammalian species exposed to cadmium respond with the induction of a low molecular weight, heat stable protein, metallothionein (MT), that binds cadmium (Cd). Metallothionein has never been isolated nor characterized in planaria. It was hypothesized that planaria should produce MT like mammalian species. Furthermore, it was anticipated that planarian MT would reduce Cd-induced DNA damage. Following exposure to sub-lethal doses of Cd, planarian homogenate from both zinc (Zn) pre-treated and non pre-treated worms was centrifuged, heat-treated and then separated with gel chromatography. Separated proteins samples were then subjected to spectrophotometric and atomic absorption analysis (AA) to identify a low molecular mass (about 7,400 daltons) Cd-binding protein with high 254 nm and low 280 nm absorbance. The protein was then further purified by diethylaminoethylcellulose (DEAE) ion exchange high-performance liquid chromatography (HPLC). Amino acid analysis showed that purified Cd binding protein contained about 28% cysteine. Planarian MT was shown to be inducible following exposure to metals, radiation and hydroperoxides.; Control and Zn pre-treated planaria were exposed to different concentrations of Cd ranging from 100 to 2,500 μg/L for different time periods. DNA fragmentation was assessed with a modified alkaline agarose gel electrophoresis assay and quantified using densitometric analysis of the migration distance. Decreases of 2.4- to 2.6-fold in electrophoretic DNA migration in Cd exposed worms were accompanied by 2.5- to 4.5-fold increases of planarian MT levels due to zinc treatment. Glutathione (GSH) depletion before Cd exposure increased DNA fragmentation in planaria. Ionizing radiation, 1.0 to 6.0 Gy, caused less DNA fragmentation in planaria pre-treated with Zn and the reduced damage was accompanied with 12.8- and 14.4-fold increases in planarian MT levels compared to control planaria. In summary, the planarian, D. dorotocephala, produces a low molecular weight heat stable protein that binds Cd. Furthermore, the protein was shown to be inducible with exposure to metals, hydroperoxides and radiation. Furthermore, planarian MT significantly reduced cadmium- and radiation-initiated DNA fragmentation.